The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens.

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Towards achieving a modern science-based paradigm for agrochemical carcinogenicity assessment.

The rodent cancer bioassay has been the standard approach to fulfill regulatory requirements for assessing human carcinogenic potential of agrochemicals, food additives, industrial chemicals, and pharmaceuticals. Decades of research have described the limitations of the rodent cancer bioassay leading to international initiatives to seek alternatives and establish approaches that modernize carcinogenicity assessment. Biologically relevant approaches can provide mechanistic information and increased efficiency for evaluating hazard and risk of chemical carcinogenicity to humans. The application of human-relevant mechanistic understanding to support new approaches to carcinogenicity assessment will be invaluable for regulatory decision-making. The present work outlines the challenges and opportunities that authorities should consider as they come together to build a roadmap that leads to global acceptance and incorporation of fit-for-purpose, scientifically defensible new approaches for human-relevant carcinogenicity assessment of agrochemicals.

Updated systematic assessment of human, animal and mechanistic evidence demonstrates lack of human carcinogenicity with consumption of aspartame.

Aspartame has been studied extensively and evaluated for its safety in foods and beverages yet concerns for its potential carcinogenicity have persisted, driven primarily by animal studies conducted at the Ramazzini Institute (RI). To address this controversy, an updated systematic review of available human, animal, and mechanistic data was conducted leveraging critical assessment tools to consider the quality and reliability of data. The evidence base includes 12 animal studies and >40 epidemiological studies reviewed by the World Health Organization which collectively demonstrate a lack of carcinogenic effect. Assessment of >1360 mechanistic endpoints, including many guideline-based genotoxicity studies, demonstrate a lack of activity associated with endpoints grouped to key characteristics of carcinogens. Other non-specific mechanistic data (e.g., mixed findings of oxidative stress across study models, tissues, and species) do not provide evidence of a biologically plausible carcinogenic pathway associated with aspartame. Taken together, available evidence supports that aspartame consumption is not carcinogenic in humans and that the inconsistent findings of the RI studies may be explained by flaws in study design and conduct (despite additional analyses to address study limitations), as acknowledged by authoritative bodies.

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

In Silico Methods for Carcinogenicity Assessment.

Screening compounds for potential carcinogenicity is of major importance for prevention of environmentally induced cancers. A large sequence of predictive models, ranging from short-term biological assays (e.g., mutagenicity tests) to theoretical models, has been attempted in this field. Theoretical approaches such as (Q)SAR are highly desirable for identifying carcinogens, since they actively promote the replacement, reduction, and refinement of animal tests. This chapter reports and describes some of the most noted (Q)SAR models based on human expert knowledge and statistical approaches, aiming at predicting the carcinogenicity of chemicals. Additionally, the performance of the selected models has been evaluated, and the results are interpreted in details by applying these predictive models to some pharmaceutical molecules.

Employing an adverse outcome pathway framework for weight-of-evidence assessment with application to the ICH S1B guidance addendum.

Across industry, there is a paradigm shift occurring for carcinogenicity testing, with the focus moving from long term animal studies to alternative approaches. Based on the explorative work done in recent years, the International Council for Harmonization (ICH) recently published a draft addendum to the S1B guidance, which allows for a weight-of-evidence (WoE) assessment to be conducted based on data gathered throughout the pharmaceutical development process and literature to mitigate some testing in rodents if the body of evidence clearly shows undertaking an animal lifetime study would not add value to the risk assessment. While several alternative approaches already exist, and other new approach methodologies (NAMs) are being explored, all of which can contribute to this WoE, it is important that all the evidence can be combined in a meaningful and consistent way to reach a conclusion. Adverse outcome pathways have been advocated as a framework for organising evidence in an integrated approach to testing and assessment, which gives context to data and can aid reaching a conclusion as to the adverse outcome (AO). This approach can be combined with a reasoning methodology to give a prediction for an AO and applied to the factors which need to be considered for the ICH S1B WoE to predict for carcinogenicity. Using this approach to the WoE assessment, consistent, scientifically robust, and transparent calls can be made as to whether conducting an animal carcinogenicity study would add value to a human risk assessment and mitigate the need to run animal studies unnecessarily.

A regulatory relic: After 60 years of research on cancer risk, the Delaney Clause continues to keep us in the past.

The Delaney Clause of the Federal Food, Drug, and Cosmetic Act became law in 1958 because of concerns that potentially harmful chemicals were finding their way into foods and causing cancer. It states, "[n]o additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal." The United States Food and Drug Administration (US FDA) and United States Environmental Protection Agency (US EPA, prior to implementation of the Food Quality Protection Act) were charged with implementing this clause. Over 60 years, advances in cancer research have elucidated how chemicals induce cancer. Significant advancements in analytical methodologies have allowed for accurate and progressively lower detection limits, resulting in detection of trace amounts. Based on current scientific knowledge, there is a need to revisit the Delaney Clause's utility. The lack of scientific merit to the Delaney Clause was very apparent when recently the US FDA had to revoke the food additive approvals of 6 synthetic flavoring substances because high dose testing in animals demonstrated a carcinogenic response. However, US FDA determined that these 6 synthetic flavoring substances do not pose a risk to public health under the conditions of intended use. The 7th substance, styrene, was de-listed because it is no longer used by industry. The scientific community is committed to improving public health by promoting relevant science in risk assessment and regulatory decision making, and this was discussed in scientific sessions at the American Association for the Advancement of Science (AAAS) 2020 Annual Meeting and the Society of Toxicology (SOT) 2019 Annual Meeting. Expert presentations included advances in cancer research since the 1950s; the role of the Delaney Clause in the current regulatory paradigm with a focus on synthetic food additives; and the impact of the clause on scientific advances and regulatory decision making. The sessions concluded with panel discussions on making the clause more relevant based on 21st-century science.

Rethinking agrochemical safety assessment: A perspective.

Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

An evolution of risk assessment for potential carcinogens in food: Scientific session proceedings.

Modern perspectives on the risk assessment of carcinogenic potential of chemicals have taken shape within the last two decades. This has been due to both developments in the understanding of the biology and etiology of cancer and by advances in in silico and in vitro assays. Moving away from a conventional binary carcinogen/non-carcinogen model, modern frameworks offer more nuanced classification structures based on the understanding of mechanisms involved or potentially involved in rodent carcinogenicity. Given these developments, a scientific session at the 2020 Winter Meeting of the Toxicology Forum was organized to explore the impact these innovative approaches will have on food safety assessments and what considerations should be addressed in developing a new carcinogenic risk assessment approach for substances in foods. The session reviewed challenges faced by food toxicologists and risk assessors, current standard approaches for evaluating carcinogenic risk of food substances, limitations of these standard approaches, and potential methods to implement next generation assays and modern carcinogenic frameworks into food safety assessments. Current perspectives of US regulatory, industry, and academic stakeholders were represented during speaker presentations and a moderated Panel Discussion. This Workshop Report provides an overview of key themes and information presented during the session. Summary statements were prepared by the authors and reviewed by the presenters but do not necessarily represent the position or policy of the FDA, the EPA, or other affiliations.

Quantitative skin exposure assessment of metals: A case study.

This article provides guidance into the quantitative risk assessment of skin exposures to metals. The use of wipe sampling methodologies has been shown to be standardised and effective for skin exposure assessment to metals. However, there is a lack of guidance documents and frameworks available to evaluate the level of health risk to workers from skin exposures to metals. Adverse health effects from exposures to metals have been described in the literature (Fernández-Nieto et al. 2006; Herman et al. 2006; Kreiss et al. 1996). Monitoring of workplace exposures typically focuses on the assessment of respiratory exposures. To provide a safe workplace there is a need to ensure all routes of exposure are risk assessed and controlled. The goal of this article was to develop skin (surface) exposure limits to metals, using the construction industry as a test environment, to quantitatively assess worker health risk of skin exposures to metals. This research concluded it was not feasible to establish single quantitative skin exposure limits to metals due to the many assumptions surrounding dermal exposures. A range of acceptable exposure limits are presented.

Integration of data across toxicity endpoints for improved safety assessment of chemicals: the example of carcinogenicity assessment.

In view of the need to enhance the assessment of consumer products called for in the EU Chemicals Strategy for Sustainability, we developed a methodology for evaluating hazard by combining information across different systemic toxicity endpoints and integrating the information with new approach methodologies. This integrates mechanistic information with a view to avoiding redundant in vivo studies, minimising reliance on apical endpoint tests and ultimately devising efficient testing strategies. Here, we present the application of our methodology to carcinogenicity assessment, mapping the available information from toxicity test methods across endpoints to the key characteristics of carcinogens. Test methods are deconstructed to allow the information they provide to be organised in a systematic way, enabling the description of the toxicity mechanisms leading to the adverse outcome. This integrated approach provides a flexible and resource-efficient means of fully exploiting test methods for which test guidelines are available to fulfil regulatory requirements for systemic toxicity assessment as well as identifying where new methods can be integrated.

Assessing chemical carcinogenicity: hazard identification, classification, and risk assessment. Insight from a Toxicology Forum state-of-the-science workshop.

The Toxicology Forum convened an international state-of-the-science workshop Assessing Chemical Carcinogenicity: Hazard Identification, Classification, and Risk Assessment in December 2020. Challenges related to assessing chemical carcinogenicity were organized under the topics of (1) problem formulation; (2) modes-of-action; (3) dose-response assessment; and (4) the use of new approach methodologies (NAMs). Key topics included the mechanisms of genotoxic and non-genotoxic carcinogenicity and how these in conjunction with consideration of exposure conditions might inform dose-response assessments and an overall risk assessment; approaches to evaluate the human relevance of modes-of-action observed in rodent studies; and the characterization of uncertainties. While the scientific limitations of the traditional rodent chronic bioassay were widely acknowledged, knowledge gaps that need to be overcome to facilitate the further development and uptake of NAMs were also identified. Since one single NAM is unlikely to replace the bioassay, activities to combine NAMs into integrated approaches for testing and assessment, or preferably into defined approaches for testing and assessment that include data interpretation procedures, were identified as urgent research needs. In addition, adverse outcome pathway networks can provide a framework for organizing the available evidence/data for assessing chemical carcinogenicity. Since a formally accepted decision tree to guide use of the best and most current science to advance carcinogenicity risk assessment is currently unavailable, a Decision Matrix for carcinogenicity assessment could be useful. The workshop organizers developed and presented a decision matrix to be considered within a carcinogenicity hazard and risk assessment that is offered in tabular form.

A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals.

Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.

The Assessment of Longitudinal Sections of Rat Female Reproductive Tissues for NTP 2-Year Toxicity and Carcinogenicity Studies.

The National Toxicology Program (NTP) now uses an extended longitudinal sectioning protocol for the uterus to better evaluate female rodent reproductive tract toxicity for all developmental and reproductive toxicology and 2-year toxicity and carcinogenicity bioassays. The previous protocol for toxicity/carcinogenicity studies involved 1 cross section midway through each uterine horn and collection of uterine cervix and vagina only if gross lesions were present. Here we compare the histological findings of the original cross sections with the additional longitudinal sections of residual uterine tissues of 7 chronic NTP rat bioassays. The goal of this study was to determine whether there might be any advantages to examining additional uterine tissue. The longitudinal protocol allowed for 10 to 20 times more uterine tissue for evaluation. Results indicate that the potential advantages of a more complete evaluation of female reproductive tract tissue include increased detection of reproductive targets, increased detection of neoplastic and nonneoplastic lesions, improved detection of tissue origin of neoplasms, less reliance on gross identification of lesions, improved accuracy in the application of severity grades, and increased detection of preneoplastic or subtle lesions.

Analysis of heavy metal content in edible honeysuckle (Lonicera japonica Thunb.) from China and health risk assessment.

Honeysuckle is a commonly used medicine for health care and treatment. To detect heavy metal pollution in honeysuckle from China and quantify the health risk of heavy metal via dietary intake, the Pb, Cd, Cr, As, Hg, Ni, Mn, Cu, and Zn contents in honeysuckle samples were determined by ICP-MS. The dissolution rate of heavy metals in honeysuckle was measured by decoction and soaking. The hazard quotient (HQ) and total hazard index (HI) were used to evaluate the noncarcinogenic risk of nine heavy metals in honeysuckle, and the carcinogenic risks of Cd and As were evaluated using the carcinogen risk. Cd exhibited the maximum permissive limit standard-exceeding rate (40.2%) in honeysuckle, followed by Cu (37.6%) and Pb (8.5%). As and Hg did not exceed the standard values, and Cr, Ni, Mn, and Zn had no limits. In a decoction fluid after 30 min of boiling, the transfer rates of Pb, Cd, As, Ni, Mn, Cu, and Zn ranged from 11.9% to 19.9%, whereas that of Cr was low (1.0%). In a soaking fluid, the transfer rates ranged from 17.0% to 56.9%; no transfer rate was detected for Hg in neither the decoction fluid nor the soaking fluid. In addition, the 95th percentile Rs of As and Cd in honeysuckle were 5.93 × 10-6 and 8.12 × 10-5, respectively. The carcinogenic risk of Cd at 56.99th percentile reached the threshold set by the World Health Organization (1.0 × 10-5). The results showed that intake of Pb, Cd, Cr, As, Hg, Ni, Mn, Cu, and Zn by the human body through honeysuckle could not cause noncarcinogenic damage. The element As had no carcinogenic risk, but Cd had a carcinogenic risk to a certain extent.

Mode of action-based risk assessment of genotoxic carcinogens.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as "omics" approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.